Assay Development & Screening Technology (ADST) group is designed to advance therapeutic development through research and development of innovative assay (test) designs and chemical library screenings. CMT disease is a collection of inherited peripheral neuropathies with diverse genetic causes resulting in motor/sensory abnormalities, chronic fatigue/pain, and adverse impacts particularly on distal limb function. CMT is generally classified as primarily demyelinating (CMT1) or axonal (CMT2), and this disorder is one of the most common inherited diseases of the nervous system. With financial support from the CMTA and in collaboration with CMT investigators we are designing and functionally validating novel assays for use in early stage translational research. CMT Type 1A (Collaboration with J. Svaren). More than half of the genetically diagnosed cases of CMT are caused by a chromosomal duplication affecting a critical myelin gene, Peripheral Myelin Protein 22 (PMP22). Since increased expression levels of PMP22 cause this neuropathy (classified as CMT1A), the simplest strategy for treatment is to achieve a relatively subtle (<2-fold) change in PMP22 regulation. Proof-of-principle studies have shown that reducing PMP22 levels leads to beneficial effects in rodent models of CMT1A. Therefore, we have developed novel assays for small molecule screening to identify compounds that effectively lower PMP22 expression and treat the root cause of CMT1A. We have designed and functionally validated novel assays for use in quantitative high-throughput screening (qHTS) that accurately reflect the physiological regulation of the PMP22 gene. This work follows an iterative design-build-test model enabling integration of advances in assay technology with key aspects of the disease physiology to achieve state-of-the-art bioassays compatible with ultra-high throughput testing platforms. Our initial assay designs using PMP22 regulatory elements driving expression of reporter genes as a surrogate of PMP22 gene expression. Second generation assays now utilize the groundbreaking approach of genome editing to insert reporters at the endogenous PMP22 locus, which allows physiological regulation of the reporter in the native chromatin environment. The assays created in this manner will enable researchers to identify both transcriptional and post-transcriptional (e.g., miRNA-mediated) effects. One of these assays is now in use at the pharmaceutical company, Sanofi-Genzyme in a parallel effort to identify novel chemical starting points for a CMT1A therapeutic. In subsequent designs, we incorporate a coincidence biocircuit reporter, developed in our laboratory to increase the fidelity of the assays by greatly minimizing the selection of reporter-specific inhibitors that can, depending on the chemical library composition, outnumber and confound selection of candidate compounds of interest. To achieve the ultimate goal of identifying small molecules that can therapeutically reduce PMP22 levels in treatment of CMT1A, we participated in an R21-funded project with an aim to perform a qHTS of the extensive molecular libraries small molecule resources at NCATS. We now are following up with the finding from a large-scale qHTS. CMT Type 1B (Collaboration with M. Shy, L. Wrabetz, J. Svaren). CMT1B, the second most common forms of the demyelinating neuropathies and can result from any one of 200 mutations in the Myelin Protein Zero (MPZ) gene. The severity of the demyelination, time and rate of progression and clinical outcome can vary widely, depending in part on the specific mutation. In a frequently occurring mechanism, the mutant MPZ activates a cellular stress response process known as the Unfolded Protein Response (UPR) that leads to demyelination in the myelin-producing Schwann cell. Suppressing a mutant MPZ-dependent pathogenic UPR activation is the primary goal of this project. We are designing and validating novel assays for use in qHTS that model mutant MPZ-dependent pathogenic UPR activation. There are three signaling branches associated with the UPR which include PERK-P/eIF2-P, ATF6, and IRE1-XBP1 and their associated downstream effector pathways, translation suppression, CHOP transcription-dependent apoptosis, UPR target gene transcription, and Endoplasmic-reticulum-associated protein degradation (ERAD), respectively. Following our iterative design-build-test model we are developing qHTS-compatible assays for each of these UPR branches. CMT Type 2A (collaboration with J. Svaren, B. Baloh). The most common form of axonal peripheral neuropathy, CMT2A results from mutations in the mitofusin 2 (Mfn2) gene. Following a 2010 CMTA-sponsored meeting of peripheral nerve and drug discovery experts principal objectives for a translational program for CMT2A included creating phenotypic cell-based assays that can be used in high-throughput screening to identify compounds that can be developed to treat CMT2A and aid in the identification of modifier genes of the primary defect that may represent alternate drug targets. We are developing an Mfn1 locus-targeted coincidence reporter biocircuit for qHTS. The assay will be used to identify compounds that can increase the level of Mfn1, a potentially compensatory mitofusin to mutated Mfn2 found in CMT2A. The assay will also be used to identify genes that when silenced cause enhance Mfn1 expression. Together the chemical screening and gene silencing studies may suggest viable drug targets to compensate for the loss of Mfn2.